The mucosal epithelium is a primary barrier against invasive microbes, so understanding the physicochemical forces involved in mucosal immune surveillance may be critical to understanding pathogenesis versus immunity. Mucosal M cells overlying lymphoid tissues are central to immune surveillance, using mechanisms for active uptake of mucosal pathogens. Ironically, many invasive pathogens ranging from Salmonella to Anthrax are also adept at using M cells for entry across the epithelial barrier. How is this uptake mediated? Recently, we showed that low ionic strength in the liquid of the airway lumen significantly enhanced particle uptake by M cells. Low ionic strength should increase long-range electrostatic interactions and increase the repulsion of negatively charged particles from negatively charged epithelium; curiously, we found a paradoxically increased uptake at M cell apical membranes in vivo. This finding has given rise to our working hypothesis: in contrast to neighboring mucosal epithelial cells, the M cell is electrostatically optimized for interaction wit microparticles in suspension as an emergent property of its smooth apical membrane. The differences in surface charge establishes an electrostatic field at the boundary between enterocytes and M cells that helps draw negatively charged particles to the M cell apical membrane. We propose two aims to test this hypothesis: (1) We will use cell culture models and a modified tangential flow chamber to model the binding of particles at the apical surface of the epithelial cells with different surface charge characteristics. We will compare fluorescent microbes and PLGA microparticles produced with different surface zeta potentials to assess binding to enterocyte versus M cell-like smooth apical membranes. (2) Using mouse models of M cell deficiency, we will study the effect of particle surface charge on uptake in the intestine, and the effect of electrostatic forces on M cell- dependent versus -independent particle uptake to immune tissues. An understanding of the effect of these electrostatic forces in the intestine will be important in defining the mechanisms of M cell mucosal immune surveillance. In addition, these forces would be expected to be important in the pathogenesis of invasive microbes such as Salmonella. Finally, our studies will help define requirements for designing mucosal vaccine delivery systems, whether designed for M cell targeted uptake or for nonspecific adhesion and M cell-independent uptake. PUBLIC HEALTH RELEVANCE: Electrostatic forces and M cell uptake at mucosal surfaces Narrative: The mucosal epithelium is a primary barrier against invasive microbes, so understanding the physicochemical forces involved in mucosal immune surveillance may be critical to understanding pathogenesis versus immunity. Mucosal M cells overlying lymphoid tissues are central to immune surveillance, using mechanisms for active uptake of mucosal pathogens. Ironically, many invasive pathogens ranging from Salmonella to Anthrax are also adept at using M cells for entry across the epithelial barrier. Recently, we discovered evidence that charge interactions between particles such as bacteria and the intestinal epithelium may be a major factor in M cell uptake. The charge interactions between bacteria and intestinal epithelium may be modulated by the surface features of M cells, which are smoother than the neighboring intestinal epithelium. That is, the local differences between M cells and intestinal epithelium may set up charge effects that drive particles to the M cell membrane. If true, this would be a striking and surprising example of how biological processes of cellular differentiation can modify local physico-chemical properties. These effects appear to be designed for optimal interactions with particles with charge properties specific to viruses and bacteria, and would appear to complement the array of innate immune receptors for detecting pathogens. We propose to test these charge effects using well-defined cell culture models to mimic M cell charge properties, and animal models with specific M cell defects. An understanding of the effect of these forces in the intestine will be important in defining the mechanisms of M cell mucosal immune surveillance. In addition, these forces would be expected to be important in the pathogenesis of invasive microbes such as Salmonella. Finally, our studies will help define requirements for designing mucosal vaccine delivery systems, whether designed for M cell targeted uptake or for nonspecific adhesion and M cell-independent uptake.